Summary This project has two broad objectives. The ?rst is to discover the rules by which genes are co-regulated by mul- tiple enhancers in complex mammalian loci. Multiple enhancers produce non-additive responses by interfering with each other's function. The project seeks to uncover the epigenetic and chromatin-structure basis of enhancer interference. The second objective is to develop a new class of mathematical models capable of simulating the regulation of multi-enhancer loci by simulating enhancer interference resulting from enhancer-promoter looping. The studies will utilize Cebpa, which encodes a transcription factor necessary for neutrophil development, as a model system. The approach combines aspects of synthetic biology, functional genomics, and mathematical modeling. In Aim 1, several transgenic myeloid cell lines, each carrying a variant of a synthetic locus in a heterol- ogous genomic location, will be constructed. The synthetic loci will be composed of the Cebpa promoter and two enhancers, whose strength will be varied by altering their sequence composition with the aid of a predictive math- ematical model of gene regulation. The response of the co-regulated gene to varying combinations of enhancer inputs will be measured to ascertain the rules of co-regulation. The hypothesis that enhancers interfere with each other via chromatin looping will be tested using chromosome conformation capture and the measurement of Mediator/Cohesin occupancy at the regulatory elements. An alternative hypothesis, that enhancers interfere by directly modifying the epigenetic state of other enhancers, will be tested by pro?ling chromatin accessibility and nucleosome positioning. These experiments will allow a causal analysis of enhancer interference by measuring alterations in the epigenetic state of one enhancer as a result of mutations introduced into another. Aim 2 is to build a mathematical model that can predict the expression of a two-enhancer locus. The proposed model will explicitly incorporate chromatin looping using a statistical mechanical framework and the chromatin conformation data acquired in Aim 1. The synthetic biology approach of Aim 1 will be complemented in Aim 3 by investigating enhancer interference in the endogenous Cebpa locus. Whether Cebpa enhancers modify the global chromatin conformation of the locus will be determined by editing an enhancer using CRISPR/Cas9 and pro?ling 3D chro- matin architecture and epigenetic state. Success in these objectives will help advance our understanding of mis-regulation of genes during oncogenesis and, in the long-term, allow us to computationally predict aberrant gene expression patterns driven by mutated DNA sequence.